, Volume 127, Issue 1, pp 29–43 | Cite as

A proteomic portrait of dinoflagellate chromatin reveals abundant RNA-binding proteins

  • Mathieu Beauchemin
  • David MorseEmail author
Original Article


Dinoflagellate chromatin is unique among eukaryotes, as the chromosomes are permanently condensed in a liquid crystal state instead of being packed in nucleosomes. However, how it is organized is still an unsolved mystery, in part due to the lack of a comprehensive catalog of dinoflagellate nuclear proteins. Here, we report the results of CHromatin Enrichment for Proteomics (CHEP) followed by shotgun mass spectrometry sequencing of the chromatin-associated proteins from the dinoflagellate Lingulodinum polyedra. Our analysis identified proteins involved in DNA replication and repair, transcription, and mRNA splicing, and showed a low level of contamination by proteins from other organelles. A limited number of proteins containing DNA-binding domains were found, consistent with the lack of diversity of these proteins in dinoflagellate transcriptomes. However, the number of proteins containing RNA-binding domains was unexpectedly high supporting a potential role for this type of protein in mediating gene expression and chromatin organization. We also identified a number of proteins involved in chromosome condensation and cell cycle progression as well as a single histone protein (H4). Our results provide the first detailed look at the nuclear proteins associated with the unusual chromatin structure of dinoflagellate nuclei and provide important insights into the biochemical basis of its structure and function.


Dinoflagellate Nucleic acid-binding protein Chromatin Proteomics 



Proteomics analyses were performed by the Center for Advanced Proteomics Analyses, a Node of the Canadian Genomic Innovation Network that is supported by the Canadian Government through Genome Canada. Computer-intensive analyses were made on the supercomputer Guillimin at McGill University, managed by Calcul Québec and Compute Canada. The operation of this supercomputer is funded by the Canada Foundation for Innovation (CFI), the ministère de l’Économie, de la science et de l’innovation du Québec (MESI), and the Fonds de recherche du Québec - Nature et technologies (FRQNT). We are grateful to Dr. F. van Dolah for the anti-PCNA antibody. We thank Drs. Annie Angers, James Omichinski, and William Zerges for their extensive review of this manuscript.


This study was funded by the National Science and Engineering Research Council of Canada (NSERC) through an Alexander-Graham-Bell Canada Doctoral Scholarship awarded to M. B. and an NSERC Discovery research grant awarded to D. M. (number 171382-03).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no competing interests.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

412_2017_643_MOESM1_ESM.pdf (11 kb)
ESM 1 Supplementary Table 1: Number of peptides and proteins identified in nine chromatin-enriched samples (PDF 11.4 kb)
412_2017_643_MOESM2_ESM.pdf (372 kb)
ESM 2 Supplementary Table 2: List of 1245 proteins identified in the chromatin enriched sample with BLAST description, GO terms and manually classified functional characterization (PDF 372 kb)
412_2017_643_MOESM3_ESM.pdf (26 kb)
ESM 3 Supplementary Table 3: Gene ontology terms significantly enriched and depleted in the chromatin enriched samples (PDF 26 kb)
412_2017_643_MOESM4_ESM.pdf (13 kb)
ESM 4 Supplementary Table 4: Proteins involved in DNA repair and processing (PDF 13 kb)
412_2017_643_MOESM5_ESM.pdf (32 kb)
ESM 5 Supplementary Table 5: Differentially expressed proteins between LD6 and LD18 (PDF 31 kb)


  1. Adhvaryu K, Firoozi G, Motavaze K, Lakin-Thomas P (2016) PRD-1, a component of the circadian system of Neurospora crassa, is a member of the DEAD-box RNA helicase family. J Biol Rhythm 31:258–271CrossRefGoogle Scholar
  2. Adl SM, Simpson AGB, Lane CE, Lukeš J, Bass D, Bowser SS, Brown MW, Burki F, Dunthorn M, Hampl V et al (2012) The revised classification of eukaryotes. J Eukaryot Microbiol 59:429–514CrossRefPubMedPubMedCentralGoogle Scholar
  3. Allada R, Meissner RA (2005) Casein kinase 2, circadian clocks, and the flight from mutagenic light. Mol Cell Biochem 274:141–149CrossRefPubMedGoogle Scholar
  4. Aranda, M., Li, Y., Liew, Y.J., Baumgarten, S., Simakov, O., Wilson, M.C., Piel, J., Ashoor, H., Bougouffa, S., Bajic, V.B., et al. (2016). Genomes of coral dinoflagellate symbionts highlight evolutionary adaptations conducive to a symbiotic lifestyle 6, 39734Google Scholar
  5. Bachvaroff TR, Place AR (2008) From stop to start: tandem gene arrangement, copy number and trans-splicing sites in the dinoflagellate Amphidinium carterae. PLoS One 3:e2929CrossRefPubMedPubMedCentralGoogle Scholar
  6. Bancel E, Bonnot T, Davanture M, Branlard G, Zivy M, Martre P (2015) Proteomic approach to identify nuclear proteins in wheat grain. J Proteome Res 14:4432–4439CrossRefPubMedGoogle Scholar
  7. Bayer T, Aranda M, Sunagawa S, Yum LK, Desalvo MK, Lindquist E, Coffroth MA, Voolstra CR, Medina M (2012) Symbiodinium transcriptomes: genome insights into the dinoflagellate symbionts of reef-building corals. PLoS One 7:e35269CrossRefPubMedPubMedCentralGoogle Scholar
  8. Beauchemin M, Roy S, Daoust P, Dagenais-Bellefeuille S, Bertomeu T, Letourneau L, Lang BF, Morse D (2012) Dinoflagellate tandem array gene transcripts are highly conserved and not polycistronic. Proc Natl Acad Sci U S A 109:15793–15798CrossRefPubMedPubMedCentralGoogle Scholar
  9. Beauchemin, M., Roy, S., Pelletier, S., Averback, A., Lanthier, F., and Morse, D. (2016). Characterization of two dinoflagellate cold shock domain proteins mSphere 1Google Scholar
  10. Bodansky S, Mintz LB, Holmes DS (1979) The mesokaryote Gyrodinium cohnii lacks nucleosomes. Biochem Biophys Res Commun 88:1329–1336CrossRefPubMedGoogle Scholar
  11. Boutet E, Lieberherr D, Tognolli M, Schneider M, Bansal P, Bridge AJ, Poux S, Bougueleret L, Xenarios I (2016) UniProtKB/Swiss-Prot, the manually annotated section of the UniProt KnowledgeBase: how to use the entry view. Methods Mole Biol (Clifton, NJ) 1374:23–54CrossRefGoogle Scholar
  12. Brunelle SA, Van Dolah FM (2011) Post-transcriptional regulation of S-phase genes in the dinoflagellate, Karenia brevis. J Eukaryot Microbiol 58:373–382CrossRefPubMedGoogle Scholar
  13. Calo E, Flynn RA, Martin L, Spitale RC, Chang HY, Wysocka J (2015) RNA helicase DDX21 coordinates transcription and ribosomal RNA processing. Nature 518:249–253CrossRefPubMedGoogle Scholar
  14. Camacho C, Coulouris G, Avagyan V, Ma N, Papadopoulos J, Bealer K, Madden TL (2009) BLAST+: architecture and applications. BMC Bioinf 10:421CrossRefGoogle Scholar
  15. Chan YH, Kwok AC, Tsang JS, Wong JT (2006) Alveolata histone-like proteins have different evolutionary origins. J Evol Biol 19:1717–1721CrossRefPubMedGoogle Scholar
  16. Chudnovsky Y, Li JF, Rizzo PJ, Hastings JW, Fagan TF (2002) Cloning, expression, and characterization of a histone-like protein from the marine dinoflagellate Lingulodinium polyedrum (dinophyceae). J Phycol 38:543–550CrossRefGoogle Scholar
  17. Costas E, Goyanes V (2005) Architecture and evolution of dinoflagellate chromosomes: an enigmatic origin. Cytogen Genome Res 109:268–275CrossRefGoogle Scholar
  18. Dagenais-Bellefeuille S, Bertomeu T, Morse D (2008) S-phase and M-phase timing are under independent circadian control in the dinoflagellate Lingulodinium. J Biol Rhythm 23:400–408CrossRefGoogle Scholar
  19. Emerson JM, Bartholomai BM, Ringelberg CS, Baker SE, Loros JJ, Dunlap JC (2015) Period-1 encodes an ATP-dependent RNA helicase that influences nutritional compensation of the Neurospora circadian clock. Proc Natl Acad Sci 112:15707–15712PubMedPubMedCentralGoogle Scholar
  20. Gornik SG, Ford KL, Mulhern TD, Bacic A, McFadden GI, Waller RF (2012) Loss of nucleosomal DNA condensation coincides with appearance of a novel nuclear protein in dinoflagellates. Curr Biol 22:2303–2312CrossRefPubMedGoogle Scholar
  21. Gotz S, Garcia-Gomez JM, Terol J, Williams TD, Nagaraj SH, Nueda MJ, Robles M, Talon M, Dopazo J, Conesa A (2008) High-throughput functional annotation and data mining with the Blast2GO suite. Nucleic Acids Res 36:3420–3435CrossRefPubMedPubMedCentralGoogle Scholar
  22. Guillard RRL, Ryther JH (1962) Studies on marine planktonic diatoms: Cyclotella nana Hufstedt and Denotula confervacea (Cleve) Gran. Can J Microbiol 8:229–239CrossRefPubMedGoogle Scholar
  23. Guillebault D, Derelle E, Bhaud Y, Moreau H (2001) Role of nuclear WW domains and proline-rich proteins in dinoflagellate transcription. Protist 152:127–138CrossRefPubMedGoogle Scholar
  24. Guillebault D, Sasorith S, Derelle E, Wurtz JM, Lozano JC, Bingham S, Tora L, Moreau H (2002) A new class of transcription initiation factors, intermediate between TATA box-binding proteins (TBPs) and TBP-like factors (TLFs), is present in the marine unicellular organism, the dinoflagellate Crypthecodinium cohnii. J Biol Chem 277:40881–40886CrossRefPubMedGoogle Scholar
  25. Hanes SD (2015) Prolyl isomerases in gene transcription. Biochim Biophys Acta 1850:2017–2034CrossRefPubMedGoogle Scholar
  26. Hastings JW (2007) The Gonyaulax clock at 50: translational control of circadian expression. Cold Spring Harb Symp Quant Biol 72:141–144CrossRefPubMedGoogle Scholar
  27. Herzog M, Soyer MO, Daney de Marcillac G (1982) A high level of thymine replacement by 5-hydroxymethyluracil in nuclear DNA of the primitive dinoflagellate Prorocentrum micans E. Eur J Cell Biol 27:151–155PubMedGoogle Scholar
  28. Ho P, Kong K, Chan Y, Tsang JSH, Wong JTY (2007) An unusual S-adenosylmethionine synthetase gene from dinoflagellate is methylated. BMC Mol Biol 8:87CrossRefPubMedPubMedCentralGoogle Scholar
  29. Homma K, Hastings JW (1989) The s phase is discrete and is controlled by the circadian clock in the marine dinoflagellate Gonyaulax polyedra. Exp Cell Res 182:635–644CrossRefPubMedGoogle Scholar
  30. Huang CK, Shen YL, Huang LF, Wu SJ, Yeh CH, Lu CA (2016) The DEAD-box RNA helicase AtRH7/PRH75 participates in pre-rRNA processing, plant development and cold tolerance in Arabidopsis. Plant Cell Physiol 57:174–191CrossRefPubMedGoogle Scholar
  31. Indiani C, O'Donnell M (2006) The replication clamp-loading machine at work in the three domains of life. Nat Rev Mol Cell Biol 7:751–761CrossRefPubMedGoogle Scholar
  32. Janouskovec J, Gavelis GS, Burki F, Dinh D, Bachvaroff TR, Gornik SG, Bright KJ, Imanian B, Strom SL, Delwiche CF et al (2017) Major transitions in dinoflagellate evolution unveiled by phylotranscriptomics. Proc Natl Acad Sci U S A 114:E171–e180CrossRefPubMedGoogle Scholar
  33. Jantzen SG, Sutherland BJG, Minkley DR, Koop BF (2011) GO trimming: systematically reducing redundancy in large gene ontology datasets. BMC Res Notes 4:267CrossRefPubMedPubMedCentralGoogle Scholar
  34. Kanehisa M, Sato Y, Kawashima M, Furumichi M, Tanabe M (2015) KEGG as a reference resource for gene and protein annotation. Nucleic Acids Res.
  35. Keeling PJ, Palmer JD (2008) Horizontal gene transfer in eukaryotic evolution. Nat Rev Genet 9:605–618CrossRefPubMedGoogle Scholar
  36. Kotoglou P, Kalaitzakis A, Vezyraki P, Tzavaras T, Michalis LK, Dantzer F, Jung JU, Angelidis C (2009) Hsp70 translocates to the nuclei and nucleoli, binds to XRCC1 and PARP-1, and protects HeLa cells from single-strand DNA breaks. Cell Stress Chaperones 14:391–406CrossRefPubMedGoogle Scholar
  37. Kustatscher G, Hegarat N, Wills KL, Furlan C, Bukowski-Wills JC, Hochegger H, Rappsilber J (2014a) Proteomics of a fuzzy organelle: interphase chromatin. EMBO J 33:648–664CrossRefPubMedPubMedCentralGoogle Scholar
  38. Kustatscher G, Wills KL, Furlan C, Rappsilber J (2014b) Chromatin enrichment for proteomics. Nat Protoc 9:2090–2099CrossRefPubMedPubMedCentralGoogle Scholar
  39. Levi-Setti R, Gavrilov KL, Rizzo PJ (2008) Divalent cation distribution in dinoflagellate chromosomes imaged by high-resolution ion probe mass spectrometry. Eur J Cell Biol 87:963–976CrossRefPubMedGoogle Scholar
  40. Lin S, Cheng S, Song B, Zhong X, Lin X, Li W, Li L, Zhang Y, Zhang H, Ji Z et al (2015) The Symbiodinium kawagutii genome illuminates dinoflagellate gene expression and coral symbiosis. Science 350:691–694CrossRefPubMedGoogle Scholar
  41. Lin S, Zhang H, Zhuang Y, Tran B, Gill J (2010) Spliced leader-based metatranscriptomic analyses lead to recognition of hidden genomic features in dinoflagellates. Proc Natl Acad Sci U S A 107:20033–20038CrossRefPubMedPubMedCentralGoogle Scholar
  42. Linder P, Jankowsky E (2011) From unwinding to clamping—the DEAD box RNA helicase family. Nat Rev Mol Cell Biol 12:505–516CrossRefPubMedGoogle Scholar
  43. Liu B, Lo SC, Matton DP, Lang BF, Morse D (2012) Daily changes in the phosphoproteome of the dinoflagellate Lingulodinium. Protist 163:746–754CrossRefPubMedGoogle Scholar
  44. Lyabin DN, Eliseeva IA, Ovchinnikov LP (2014) YB-1 protein: functions and regulation. Wiley Int Rev RNA 5:95–110CrossRefGoogle Scholar
  45. Marchler-Bauer A, Derbyshire MK, Gonzales NR, Lu S, Chitsaz F, Geer LY, Geer RC, He J, Gwadz M, Hurwitz DI et al (2015) CDD: NCBI’s conserved domain database. Nucleic Acids Res 43:D222–D226CrossRefPubMedGoogle Scholar
  46. Marinov GK, Lynch M (2016) Diversity and divergence of dinoflagellate histone proteins. G3: Genes|Genomes|Genetics 6:397–422CrossRefGoogle Scholar
  47. Matsuda S, Adachi J, Ihara M, Tanuma N, Shima H, Kakizuka A, Ikura M, Ikura T, Matsuda T (2016) Nuclear pyruvate kinase M2 complex serves as a transcriptional coactivator of arylhydrocarbon receptor. Nucleic Acids Res 44:636–647CrossRefPubMedGoogle Scholar
  48. Michels AA, Kanon B, Konings AWT, Ohtsuka K, Bensaude O, Kampinga HH (1997) Hsp70 and Hsp40 chaperone activities in the cytoplasm and the nucleus of mammalian cells. J Biol Chem 272:33283–33289CrossRefPubMedGoogle Scholar
  49. Montpetit B, Thomsen ND, Helmke KJ, Seeliger MA, Berger JM, Weis K (2011) A conserved mechanism of DEAD-box ATPase activation by nucleoporins and InsP6 in mRNA export. Nature 472:238–242CrossRefPubMedPubMedCentralGoogle Scholar
  50. Morey JS, Van Dolah FM (2013) Global analysis of mRNA half-lives and de novo transcription in a dinoflagellate, Karenia brevis. PLoS One 8:e66347CrossRefPubMedPubMedCentralGoogle Scholar
  51. Mouveaux T, Oria G, Werkmeister E, Slomianny C, Fox BA, Bzik DJ, Tomavo S (2014) Nuclear glycolytic enzyme enolase of Toxoplasma gondii functions as a transcriptional regulator. PLoS One 9:e105820CrossRefPubMedPubMedCentralGoogle Scholar
  52. Nassoury N, Fritz L, Morse D (2001) Circadian changes in ribulose-1,5-bisphosphate carboxylase/oxygenase distribution inside individual chloroplasts can account for the rhythm in dinoflagellate carbon fixation. Plant Cell 13:923–934CrossRefPubMedPubMedCentralGoogle Scholar
  53. Nosenko T, Bhattacharya D (2007) Horizontal gene transfer in chromalveolates. BMC Evol Biol 7:173CrossRefPubMedPubMedCentralGoogle Scholar
  54. Oakley GG, Patrick SM (2010) Replication protein A: directing traffic at the intersection of replication and repair. Front Biosci (Landmark ed) 15:883–900CrossRefGoogle Scholar
  55. Ohta S, Bukowski-Wills JC, Sanchez-Pulido L, Alves Fde L, Wood L, Chen ZA, Platani M, Fischer L, Hudson DF, Ponting CP et al (2010) The protein composition of mitotic chromosomes determined using multiclassifier combinatorial proteomics. Cell 142:810–821CrossRefPubMedPubMedCentralGoogle Scholar
  56. Plumbridge J (2001) DNA binding sites for the Mlc and NagC proteins: regulation of nagE, encoding the N-acetylglucosamine-specific transporter in Escherichia coli. Nucleic Acids Res 29:506–514CrossRefPubMedPubMedCentralGoogle Scholar
  57. Qin H, Wang Y (2009) Exploring DNA-binding proteins with in vivo chemical cross-linking and mass spectrometry. J Proteome Res 8:1983–1991CrossRefPubMedGoogle Scholar
  58. Rizzo PJ, Jones M, Ray SM (1982) Isolation and properties of isolated nuclei from the Florida red tide dinoflagellate Gymnodinium breve (Davis). J Protozool 29:217–222CrossRefPubMedGoogle Scholar
  59. Rizzo PJ, Nooden LD (1972) Chromosomal proteins in the dinoflagellate alga Gyrodinium cohnii. Science (New York, NY) 176:796–797CrossRefGoogle Scholar
  60. Robinson MD, McCarthy DJ, Smyth GK (2010) edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics (Oxf, England) 26:139–140CrossRefGoogle Scholar
  61. Roy S, Beauchemin M, Dagenais-Bellefeuille S, Letourneau L, Cappadocia M, Morse D (2014a) The Lingulodinium circadian system lacks rhythmic changes in transcript abundance. BMC Biol 12:107CrossRefPubMedPubMedCentralGoogle Scholar
  62. Roy S, Letourneau L, Morse D (2014b) Cold-induced cysts of the photosynthetic dinoflagellate Lingulodinium polyedrum have an arrested circadian bioluminescence rhythm and lower levels of protein phosphorylation. Plant Physiol 164:966–977CrossRefPubMedGoogle Scholar
  63. Roy S, Morse D (2012) A full suite of histone and histone modifying genes are transcribed in the dinoflagellate Lingulodinium. PLoS One 7:e34340CrossRefPubMedPubMedCentralGoogle Scholar
  64. Roy S, Morse D (2013) Transcription and maturation of mRNA in dinoflagellates. Microorganisms 1:71–99Google Scholar
  65. Roy S, Morse D (2014) The dinoflagellate Lingulodinium has predicted casein kinase 2 sites in many RNA binding proteins. Protist 165:330–342CrossRefPubMedGoogle Scholar
  66. Sala-Rovira M, Geraud ML, Caput D, Jacques F, Soyer-Gobillard MO, Vernet G, Herzog M (1991) Molecular cloning and immunolocalization of two variants of the major basic nuclear protein (HCc) from the histone-less eukaryote Crypthecodinium cohnii (Pyrrhophyta). Chromosoma 100:510–518CrossRefPubMedGoogle Scholar
  67. Sigee DC (1983) Structural DNA and genetically active DNA in dinoflagellate chromosomes. Bio Systems 16:203–210CrossRefPubMedGoogle Scholar
  68. Sikorskaite S, Rajamaki ML, Baniulis D, Stanys V, Valkonen JP (2013) Protocol: optimised methodology for isolation of nuclei from leaves of species in the Solanaceae and Rosaceae families. Plant Methods 9:31CrossRefPubMedPubMedCentralGoogle Scholar
  69. Singh G, Pratt G, Yeo GW, Moore MJ (2015) The clothes make the mRNA: past and present trends in mRNP fashion. Annu Rev Biochem 84:325–354CrossRefPubMedPubMedCentralGoogle Scholar
  70. Skabkin MA, Evdokimova V, Thomas AA, Ovchinnikov LP (2001) The major messenger ribonucleoprotein particle protein p50 (YB-1) promotes nucleic acid strand annealing. J Biol Chem 276:44841–44847CrossRefPubMedGoogle Scholar
  71. Sonmez C, Baurle I, Magusin A, Dreos R, Laubinger S, Weigel D, Dean C (2011) RNA 3′ processing functions of Arabidopsis FCA and FPA limit intergenic transcription. Proc Natl Acad Sci U S A 108:8508–8513CrossRefPubMedPubMedCentralGoogle Scholar
  72. Soyer M-O, Haapala OK (1974a) Electron microscopy of RNA in dinoflagellate chromosomes. Histochemistry 42:239–246CrossRefPubMedGoogle Scholar
  73. Soyer M-O, Haapala OK (1974b) Structural changes of dinoflagellate chromosomes by pronase and ribonuclease. Chromosoma 47:179–192CrossRefPubMedGoogle Scholar
  74. Soyer-Gobillard MO, Herzog M (1985) The native structure of dinoflagellate chromosomes—involvement of structural RNA. Eur J Cell Biol 36:334–342Google Scholar
  75. Taylor FJR, Hoppenrath M, Saldarriaga JF (2008) Dinoflagellate diversity and distribution. Biodivers Conserv 17:407–418CrossRefGoogle Scholar
  76. The-Gene-Ontology-Consortium (2015) Gene Ontology Consortium: going forward. Nucleic Acids Res 43:D1049–D1056CrossRefGoogle Scholar
  77. Veldhuis MJW, Cucci TL, Sieracki ME (1997) Cellular DNA content of marine phytoplankton using two new fluorochromes: taxonomic and ecological implications. J Phycol 33:527–541CrossRefGoogle Scholar
  78. Ventura M, Mateo F, Serratosa J, Salaet I, Carujo S, Bachs O, Pujol MJ (2010) Nuclear translocation of glyceraldehyde-3-phosphate dehydrogenase is regulated by acetylation. Int J Biochem Cell Biol 42:1672–1680CrossRefPubMedGoogle Scholar
  79. Vizcaino JA, Csordas A, Del-Toro N, Dianes JA, Griss J, Lavidas I, Mayer G, Perez-Riverol Y, Reisinger F, Ternent T et al (2016) 2016 update of the PRIDE database and its related tools. Nucleic Acids Res 44:11033CrossRefPubMedPubMedCentralGoogle Scholar
  80. Xiang N, He M, Ishaq M, Gao Y, Song F, Guo L, Ma L, Sun G, Liu D, Guo D et al (2016) The DEAD-box RNA helicase DDX3 interacts with NF-kappaB subunit p65 and suppresses p65-mediated transcription. PLoS One 11:e0164471CrossRefPubMedPubMedCentralGoogle Scholar
  81. Zhang J, Chen QM (2013) Far upstream element binding protein 1: a commander of transcription, translation and beyond. Oncogene 32:2907–2916CrossRefPubMedGoogle Scholar

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Authors and Affiliations

  1. 1.Institut de Recherche en Biologie Végétale, Département de Sciences BiologiquesUniversité de MontréalMontréalCanada

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